Human papillomaviruses (HPV) are non-enveloped DNA viruses that induce hyperproliferative lesions of cutaneous and mucosal epithelia (warts).(Ref: P Howleyxe2x80x94In Fields Virology 2nd Edn. Chap. 58 pp1625-1676 eds Fields et al Raven Press NY 1990) Genital HPV infection is one of the most common sexually transmitted diseases. (Ref Maw RD, Reitas M and Roy M, Int J STD+AIDS 9, 571-578, (1998)). It is estimated that visible genital warts are present in 1% of sexually active adults in the USA and that at least 15% have subclinical infection. (Koutsky L., Am. J. Med. 102, 3-8 (1997)). Over 90% of benign external warts (condyloma acuminata) are caused by HPV genotypes 6 and 11 (Handsfield H. H. Am. J. Med. 102, 16-27 (1997)).
E1 Helicase is the only known HPV enzyme and is essential for viral DNA replication. The E1 protein has been shown to possess ATPase and ATP-dependent DNA helicase catalytic activities. It is proposed to function as a hexameric helicase and sequence homology classifies it as a member of helicase superfamily III (other members: SV40 TAg, parvovirus NS1). Inhibition of the ATPase or helicase functions of this enzyme would be predicted to result in inhibition of HPV DNA replication.
The object of the present invention is to provide novel compounds which are potent inhibitors of the ATPase activity of the helicase enzyme and which accordingly show a potential to be efficacious as antiviral drugs.
The present invention comprises novel benzodiazepine derivatives of the formula 
The novel compounds are inhibitors of the human papilloma virus (HPV) E1 helicase enzyme and can therefore be used as therapeutic agents for HPV mediated diseases.
The present invention comprises novel compounds of the formula: 
wherein
R1 is H, lower alkyl, lower alkenyl, lower alkynyl, aryl lower alkyl, lower alkyl carbonyl, aryl carbonyl, lower alkyl amino carbonyl, aryl amino carbonyl, lower alkoxy carbonyl, aryloxy carbonyl,
R2a, R2b independently are H or lower alkyl or
R2a and R2b together are oxo,
R1 and R2a or R2b together with the nitrogen and the carbon atom to which they are attached form aheterocycle;
R3a, R3b independently are H or lower alkyl
R4 and R5 together with the two carbon atoms to which they are attached form aryl or a heterocycle,
R6 and R7 is H or lower alkyl and
R8 is aryl or heterocyclyl.
The term xe2x80x9clowerxe2x80x9d used in combination with alkyl and alkoxy defines an optionally substituted straight chained or branched alkyl chain carrying 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. The term xe2x80x9clowerxe2x80x9d used in combination with alkenyl and alkynyl defines an optionally substituted straight chained or branched alkenyl or alkynyl chain carrying 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.
Lower alkyl in R1,R2a, R2b, R3a, R3b, R6 and R7 accordingly preferably stands for methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and tert.-butyl.
Lower alkoxy in R1 preferably stands for methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy and tert.-butoxy.
Lower alkenyl in R1 accordingly preferably is vinyl, 1-propenyl, 2-propenyl, i-propenyl and butenyl and its isomers.
Lower alkynyl in R1 accordingly preferably is ethynyl, propynyl and its isomers and butynyl and its isomers.
Preferred meaning for R1 is methyl.
Preferred meaning for R2a, R2b, R3a, R3b, R6 and R7 is hydrogen.
Suitable substituents of an alkyl chain can be selected from one or more of aryl, heterocyclyl, carboxyl, cyano, alkoxy, cycloalkyl oxy, aryl oxy, heterocyclyl oxy, hydroxyl, alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, alkoxy carbonyl, cycloalkyl oxy carbonyl, aryl oxy carbonyl, heterocyclyl oxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, cycloalkyl amino carbonyl, aryl amino carbonyl, heterocyclyl amino carbonyl, amino, alkyl amino, dialkyl amino, alkenyl amino, alkynyl amino, cycloalkyl amino, aryl amino, heterocyclyl amino, alkyl carbonyl amino, dialkyl carbonyl amino, cycloalkyl carbonyl amino, aryl carbonyl amino, heterocyclyl carbonyl amino, alkoxy carbonyl amino, cycloalkyl oxy carbonyl amino, aryloxy carbonyl amino, heterocylyl oxy carbonyl amino, alkyl amino carbonyl amino, dialkyl amino carbonyl amino, cycloalkyl amino carbonyl amino, aryl amino carbonyl amino, heterocyclyl amino carbonyl amino alkyl sulfonyl amino, cycloalkyl sulfonyl amino, aryl sulfonyl amino, heterocyclyl sulfonyl amino, nitro, alkyl sulfonyl, cycloalkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl, thio, alkyl thio, cycloalkyl thio, aryl thio, heterocyclyl thio or halogen.
In all cases above where there are NH groups, the free hydrogen may also be substituted, preferably with lower alkyl. Examples are alkyl carbonyl (lower alkyl) amino, cycloalkyl (lower alkyl) amino carbonyl or alkoxy carbonyl (lower alkyl) amino.
The term cycloalkyl has the meaning of an optionally substituted cycloalkyl group containing 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl or adamantyl which can also be benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle or carbocycle, e.g. to phenyl.
The term aryl denotes optionally substituted phenyl and naphthyl, both optionally benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle or carbocycle e.g. to cyclohexyl or cyclopentyl.
The term heterocyclyl stands for an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle which contains one or more hetero atoms selected from nitrogen, oxygen and sulfur which can also be benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic carbocycle or heterocycle.
Preferred heterocycles are oxazolyl, isoxazolyl, furyl, tetrahydrofuryl, 1,3-dioxolanyl, dihydropyranyl, thienyl, pyrazinyl, isothiazolyl isoquinolinyl, indolyl, indazolyl, quinolinyl, dihydrooxazolyl, pyrimidinyl, benzofuranyl, tetrazolyl, pyrrolidinonyl, (N-oxide)-pyridinyl, pyrrol, triazolyl e.g. 1,2,4-triazolyl, pyrazolyl, benzotriazolyl, priperidinyl, morpholinyl, thiazolyl, pyridinyl, dihydrothiazolyl, imidazolidinyl, pyrazolinyl, benzothienyl, piperazinyl, imidazolyl, thia diazolyl e.g. 1,2,3-thiadiazolyl, and benzothiazolyl.
Suitable substituents for cycloalkyl, aryl, heterocyclyl can be selected from those named for alkyl, in addition however lower alkyl, lower alkenyl and lower alkynyl are substituents to be added to the selection.
The term halogen stands for fluorine, chlorine, bromine and iodine.
R4 and R5 together with the two carbon atoms to which they are attached preferably form optionally substituted aryl, more preferably form optionally substituted phenyl.
R8 preferably stands for optionally substituted aryl, more preferably for optionally substituted phenyl.
Any functional (i.e. reactive) group present in a side-chain may be protected, with the protecting group being a group which is known per se, for example, as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd Ed., T. W. Greene and P. G. M. Wuts, John Wiley and Sons, New York, N.Y., 1991. For example, an amino group can be protected by a tert.-butoxycarbonyl (BOC), formyl, trityl, benzyloxycarbonyl (Z), 9-fluorenylmethyloxcarbonyl (FMOC), trifluoroacetyl, 2-(biphenylyl)isopropoxycarbonyl or isobornyloxycarbonyl group or in the form of a phthalimido group; or a hydroxyl group can be protected by a tert.butyldimethylsilyl, tetrahydropyranyl, 4-methoxybenzyl, or benzyl; or a carboxyl group can be protected in the form of an ester, for example as a methyl or tert.butyl ester. The protecting group may be retained in the final compound or optionally be removed by techniques known in the art.
The compounds of this invention may contain one or more asymmetric carbon atoms and may therefore occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Furthermore, where a compound of the invention contains an olefinic double bond, this can have the (E) or (Z) configuration. Also, each chiral center may be of the R or S configuration. All such isomeric forms of these compounds are embraced by the present invention.
Compounds of formula (I) which are acidic can form pharmaceutically acceptable salts with bases such as alkali metal hydroxides, e.g. sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides, e.g. calcium hydroxide, barium hydroxide and magnesium hydroxide, and the like; with organic bases e.g. N-ethyl piperidine, dibenzylamine, and the like. Those compounds of formula (I) which are basic can form pharmaceutically acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric acid and hydrobromic acid, sulphuric acid, nitric acid and phosphoric acid, and the like, and with organic acids, e.g. with acetic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malic acid, salicylic acid, citric acid, methanesulphonic acid and p-toluene sulphonic acid, and the like. The formation and isolation of such salts can be carried out according to methods known in the art.
Preferred compounds of formula (I) are those having the formula 
wherein
R1, R2a, R2b, R3a, R3b, R6 and R 7 are as above and
R9, R10, R11, R12, R13, R14, R15, R16 and R17 independently are H, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, aryl, heterocyclyl, carboxyl, cyano, alkoxy, cycloalkyl oxy, aryl oxy, heterocyclyl oxy, hydroxyl, alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, alkoxy carbonyl, cycloalkyl oxy carbonyl, aryl oxy carbonyl, heterocyclyl oxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, cycloalkyl amino carbonyl, aryl amino carbonyl, heterocyclyl amino carbonyl, amino, alkyl amino, dialkyl amino, alkenyl amino, alkynyl amino, cycloalkyl amino, aryl amino, heterocyclyl amino, alkyl carbonyl amino, dialkyl carbonyl amino, cycloalkyl carbonyl amino, aryl carbonyl amino, heterocyclyl carbonyl amino, alkoxy carbonyl amino, cycloalkyl oxy carbonyl amino, aryloxy carbonyl amino, heterocylyl oxy carbonyl amino, alkyl amino carbonyl amino, dialkyl amino carbonyl amino, cycloalkyl amino carbonyl amino, aryl amino carbonyl amino, heterocyclyl amino carbonyl amino, alkyl carbonyl amino alkyl carbonyl amino, dialkyl amino carbonyl amino alkyl carbonyl amino, cycloalkyl carbonyl amino alkyl carbonyl amino, aryl carbonyl amino alkyl carbonyl amino, heterocyclyl carbonyl amino alkyl carbonyl amino, alkyl sulfonyl amino, cycloalkyl sulfonyl amino, aryl sulfonyl amino, heterocyclyl sulfonyl amino, nitro, alkyl sulfonyl, cycloalkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl, thio, alkyl thio, cycloalkyl thio, aryl thio, heterocyclyl thio or halogen or
R10 and R11 together with the two carbon atoms to which they are attached form optionally
substituted aryl or an optionally substituted heterocycle.
Preferred meaning for R1 is methyl.
Preferred meaning for R2a, R2b, R3a, R3b, R6 and R7 is hydrogen.
More preferred compounds of formula (I) are those having the formula 
wherein
R1, R2a, R2b, R3a, R3b, R6, R7, R9, R10, R11, R12, R13, R14, R15 and R16 are as above and wherein
X is (CH2xe2x80x94)n with n being an integer between 0 and 3, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NR23, wherein R23 is H or lower alkyl,
Y is xe2x80x94(CH2xe2x80x94)n with n being an integer between 0 and 3, and when X is (CH2xe2x80x94)n with n being an integer between 0 and 3 then Y is S, O or xe2x80x94NR23 wherein R23 is as above,
R18, R19, R20 and R22 independently are H, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, aryl, heterocyclyl, carboxyl, cyano, alkoxy, cycloalkyl oxy, aryl oxy, heterocyclyl oxy, hydroxyl, alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, alkoxy carbonyl, cycloalkyl oxy carbonyl, aryl oxy carbonyl, heterocyclyl oxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, cycloalkyl amino carbonyl, aryl amino carbonyl, heterocyclyl amino carbonyl, amino, alkyl amino, dialkyl amino, alkenyl amino, alkynyl amino, cycloalkyl amino, aryl amino, heterocyclyl amino, alkyl carbonyl amino, dialkyl carbonyl amino, cycloalkyl carbonyl amino, aryl carbonyl amino, heterocyclyl carbonyl amino, alkoxy carbonyl amino, cycloalkyl oxy carbonyl amino, aryloxy carbonyl amino, heterocylyl oxy carbonyl amino, alkyl amino carbonyl amino, dialkyl amino carbonyl amino, cycloalkyl amino carbonyl amino, aryl amino carbonyl amino, heterocyclyl amino carbonyl amino, alkyl carbonyl amino alkyl carbonyl amino, dialkyl amino carbonyl amino alkyl carbonyl amino, cycloalkyl carbonyl amino alkyl carbonyl amino, aryl carbonyl amino alkyl carbonyl amino, heterocyclyl carbonyl amino alkyl carbonyl amino, alkyl sulfonyl amino, cycloalkyl sulfonyl amino, aryl sulfonyl amino, heterocyclyl sulfonyl amino, nitro, alkyl sulfonyl, cycloalkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl, thio, alkyl thio, cycloalkyl thio, aryl thio, heterocyclyl thio or halogen.
Preferred meaning for R1 is methyl.
Preferred meaning for R2a, R2b, R3a, R3b, R6 and R7 is hydrogen.
Examples of compounds of formula I are set out below in table A:
The benzodiazepines provided by the present invention are potent inhibitors of the ATPase activity of the human papillomavirus E1 helicase enzyme. They accordingly are therapeutically active substances in the treatment of HPV mediated diseases and therefore can be used as medicaments, either alone or combined with other therapeutically active agents.
The benzodiazepines provided by the present invention are in particular useful in combating HPV disease states such as cutaneous warts on any part of the body, including palmar, plantar and flat/plane warts, anogenital warts (condylomata acuminata), including external and internal (intraurethral, vaginal and cervical) warts, all grades of CIN (cervical intraepithelial neoplasia) and SIL (squamous intraepithelial lesions), recurrent laryngeal papillomatosis (laryngeal warts), epidermodysplasia verruciformis, focal epithelial hyperplasia (Heck""s disease), warts or intraepithelial neoplasia affecting the oral and nasal cavities and conjunctival warts.
The compounds of the present invention can be prepared by coupling of a compound of formula 
wherein
R4 and R5 are as above and Hal is a halogen atom with a diamine of formula
R1NH(CH2CH2)NH2xe2x80x83xe2x80x83V
wherein R1 is as above.
The reaction can be carried out in a conventional manner known to the skilled in the art or following the adaption of a method provided in Journal of Organic Chemistry (1963) p 3013 by Sternbach et al., suitably in pyridine as solvent and at elevated temperature.
The compounds of formula IV and V are new intermediates not known to the state of the art and therefore are also subject of the present invention.
The compounds of formula IV are accessible by condensation of a ketone of formula 
wherein
R4 and R5 are as above and Hal is a halogen atom with an aldehyde of the formula
R8CHOxe2x80x83xe2x80x83VII
wherein R8 is as above following methods known to the skilled in the art.
Alternatively the compounds of the present invention can be prepared by coupling of a phosphoric acid ester of formula 
wherein
R1, R4 and R5 are as above and wherein R23 is lower alkyl with an aldehyde of the formula
R8CHOxe2x80x83xe2x80x83VII
wherein R8 is as above.
The reaction can be carried out in a conventional manner known to the skilled in the art or following the adaption of a method provided in Buzas and Finet, Tetrahedron Letters 1976, p 2433, suitably in the presence of an alkali metal hydride in tetrahydrofuran.
The phosphoric acid ester of formula VIII is accessible by a reaction of a compound of formula 
wherein
R4, R5 and R23 and Hal are as above with a diamine of the formula
R1NH(CH2CH2)NH2xe2x80x83xe2x80x83V
wherein R1 is as above.
The intermediates of formulas VII, VIII, IX and X are not known to the state of the art and are therefore also subject of the present invention.
The compound of formula IX itself can be synthesized starting from a ketone of formula VI by a conversion with a compound of formula 
wherein
R23 is as above. This process can be carried out using methods known to the skilled in the art following the adaption of a method provided in Kim et al, Journal of the Chemical Society, Perkin Transactions I 1997, pp 1361.
With regard to the starting materials that are known compounds some of these may be purchased from commercial suppliers. Other starting materials that are known and their analogues can be prepared by methods well known in the art.
For assaying E1 ATPase activity the HPV E1 enzyme has been prepared and purified as follows:
The HPV(11) E1 used in this assay is expressed as a maltose binding protein (MBP) fusion protein from SF9 cells using a baculovirus expression system. Frozen pellets of these cells are thawed by adding the pellet directly into a buffer at 4 OC containing 50 mM Tris-HCl pH 7.5, 20 mM dithiothreitol (DTT), 1 mM EDTA, 600 mM NaCl, 20% glycerol and 3 xe2x80x9cCompletexe2x80x9d protease inhibitor tablets/50 ml (Boehringer Mannheim cat.no.1 697 498). The cell suspension is then sonicated for 3xc3x9710 seconds before centrifuging at 18000 rpm for 30 minutes to remove the cell debris. The clarified extract is then passed down a DE52 ion exchange column which is washed with a buffer containing 50 mM Tris-HCl pH 7.5, 2 mM DTT, 1 mM EDTA, 600 mM NaCl and 20% glycerol. The column flow through plus the first 10 mls of wash buffer are then passed down an amylose affinity column which binds MBP tagged proteins. This column is washed with 3 column bed volumes of the wash buffer before being eluted with wash buffer containing 10 mM maltose. The eluted protein peak containing purified HPV(11) E1 is then dialysed overnight against 2 L of buffer containing 20 mM Tris-HCl pH 7.5, 2 mM DTT, 20 mM NaCl and 20% glycerol. The dialyzed material is used in the assay.
E1 ATPase activity can be measured as follows:
The standard reaction contains 50 mM MOPs KOH pH 7.0, 500 xcexcM MgCl2, 20 xcexcM ATP, 50 mM NaCl, 40 xcexcl of a suitably diluted enzyme extract and 10 xcexcl of inhibitor in a final volume of 100 xcexcl. The ATP contains 0.1 xcexcCi [xcex333P]ATP per reaction. The enzyme diluent contains 50 mM MOPs KOH pH7.0 and 1 mg/ml BSA. The inhibitor is diluted to give a range of concentrations in neat DMSO. Reaction tubes are incubated for 1 hour at 37xc2x0 C. (producing linear kinetics) after which the reaction is terminated by heat inactivation at 85xc2x0 C. for 2 minutes. 500 xcexcl 15% activated charcoal in PBS is then added to each reaction. The activated charcoal used is 100-400 mesh untreated powder (Sigma cat No C-5260); prior to use in the assay this is washed in PBS several times and allowed to settle out under gravity, any fine particles still in suspension being decanted off after each wash. The reactions are left on ice for 1 hour after which the charcoal is pelleted out by centrifugation at 14000 rpm for 10 minutes. The charcoal pellet contains unconverted [xcex333P]ATP, whilst any free inorganic 33P, the reaction product, will remain in the supernatant. The amount of radioactivity present in 300 xcexcl of supernatant is then measured by scintillation spectrophotometry.
The results can be calculated as follows:
The degree of inhibition at each inhibitor concentration is expressed as a percentage of the control reaction (100%) after subtracting a measured blank value, which represents the amount of free inorganic 33P present in a reaction containing heat inactivated enzyme. An IC50 value (concentration of test compound which inhibits enzyme activity by 50%) is then calculated from a dose response curve of log10 inhibitor concentration against percentage of the control reaction.
Preferred compounds of the invention tested in the above assay have an IC50 value up to about 50 xcexcM.
Specific examples of IC50 values for some compounds of the invention are set out in the table B below.
The compound of the present invention as well as its pharmaceutically usable acid addition salts can be used as medicaments, e.g. in the form of pharmaceutical preparations. The pharmaceutical preparations can be administered orally, e.g. in the form of tablets, coated tablets, dragxc3xa9es, hard and soft gelatine capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions or topically, e.g. in the form of a cream, a gel or a solution.
The compound of the present invention and its pharmaceutically usable acid addition salts can be processed with pharmaceutically inert, inorganic or organic excipients for the production of tablets, coated tablets, dragees and hard gelatine capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc can be used as such excipients e.g. for tablets, dragxc3xa9es and hard gelatine capsules.
Suitable excipients for soft gelatine capsules are e.g. vegetable oils, waxes, fats, semi-solid and liquid polyols etc.
Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar, glucose etc.
Suitable excipients for injection solutions are e.g. water, alcohols, polyols, glycerol, vegetable oils etc.
Suitable excipients for suppositories are e.g. natural or hardened oils, waxes, fats, semi-liquid or liquid polyols etc.
Suitable excipients for topical gels are e.g. natural gums such as xanthan and tragacanth, semisynthetic cellulose derivatives such as methylcelluloses and carboxymethylcelluloses, carbomers, clays such as silicates and presevatives such as benzoic acid or parabens.
Suitable excipients for topical creams are e.g. oils and waxes, emulsifying agents such as surfactants and polymers such as polyoxamers and preservatives.
Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 10 to 1000 mg per person of the compound of formula I should be appropriate, although the above upper limit can also be exceeded when necessary.